We used RNA sequencing to characterize gene expression of CD4+ CD8a+ double positive (DP), Foxp3+ Treg (TR) and CD4+ single positive (SP) cells in the lamina propria (LP) and intraepithelial compartment (IEL) that had differentiante from the same clonal transnuclear (TN) precursor. Overall design: We adoptively transferred CD4+ CD8a- Foxp3-GFP- isolated from pTregTN/RKO/Foxp3-GFP mice into TCRaßKO hosts. After 6 weeks, we sorted transferred CD4+ CD8a+, Foxp3+ pTreg as well as unconverted CD4+ CD8a- Foxp3-GFP- from the small intestine LP and IEL compartments for whole transcriptome analysis by mRNA sequencing.
Tissue-specific emergence of regulatory and intraepithelial T cells from a clonal T cell precursor.
Specimen part, Subject
View SamplesMedulloblastoma is the most frequent malignant pediatric brain tumor and is divided into at least four subgroups known as Wnt, SHH, Group 3 and Group 4. Here we characterized gene regulation mechanisms in the most aggressive subtype, Group 3 tumors, through genome-wide chromatin and expression profiling. Our results show that most active distal sites in these tumors are occupied by the transcription factor OTX2. Highly active OTX2 bound enhancers are often arranged as clusters of adjacent peaks and are also bound by the transcription factor NEUROD1. These sites are responsive to OTX2 and NEUROD1 knockdown and could also be generated de novo upon ectopic OTX2 expression in primary cells, showing that OTX2 cooperates with NEUROD1 and plays a major role in maintaining and possibly establishing regulatory elements as a pioneer factor. Among OTX2 target genes we identified the kinase NEK2, whose knockdown and pharmacological inhibition decreased cell viability. Our studies thus show that OTX2 controls the regulatory landscape of Group 3 medulloblastoma through cooperative activity at enhancer elements and contributes to the expression of critical target genes. Overall design: Primary Group 3 Medulloblastomas tumor samples were analyzed by RNA-seq. Group 3 medulloblastoma cell line (D341) was analyzed by RNA-seq. OTX2 was depleted by infection with lentiviral shRNAs (sh OTX2 and sh GFP control). Raw data not provided for primary Medulloblastoma samples due to patient privacy concerns. Submitter states that the raw data for these samples will be submitted to dbGaP.
OTX2 Activity at Distal Regulatory Elements Shapes the Chromatin Landscape of Group 3 Medulloblastoma.
Cell line, Subject
View SamplesTranslation initiation factor eIF4E is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. Using immortalized human breast epithelial cells, we report that elevated expression of elF4E translationally activates the TGF pathway, promoting cell invasion, loss of cell polarity, increased cell survival and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate selective translation of integrin 1 mRNA, which drives the translationally controlled assembly of a TGF receptor signaling complex containing 31 integrins, -catenin, TGF receptor I, E-cadherin and phosphorylated Smads2/3. This receptor complex acutely sensitizes non-malignant breast epithelial cells to activation by typically sub-stimulatory levels of activated TGF. TGF can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF, eIF4E confers selective mRNA translation, reprogramming non-malignant cells to an invasive phenotype by reducing the set-point for stimulation by activated TGF. Overexpression of eIF4E may be a pro-invasive facilitator of TGF activity.
Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells.
Sex, Specimen part, Cell line
View SamplesMyotonic dystrophes (DM), the most common adult muscular dystrophy, are the first recognized examples of RNA-mediated diseases in which expression of mutant RNAs containing expanded CUG or CCUG repeats interfere with the splicing of other mRNAs. Using whole-genome microarrays, we found that alternative splicing of the BIN1 mRNA is altered in DM skeletal muscle tissues, resulting in the expression of an inactive form of BIN1 deprived of phosphoinositide-binding and membrane-tubulating activities. BIN1 is involved in tubular invaginations of the plasma membrane and is essential for biogenesis of the muscle T-tubules, which are specialized skeletal muscle membrane structures essential to correct excitation-contraction (E-C) coupling. Mutations in the BIN1 gene cause centronuclear myopathy (CNM) that shares some histopathological features with DM, and both diseases are characterized by muscle weakness. Consistent with a loss-of-function of BIN1, muscle T-tubules were altered in DM patients, and membrane tubulation was restored upon expression of the correct splicing form of BIN1 in DM muscle cells. By deciphering the mechanism of BIN1 splicing mis-regulation we demonstrate that the splicing regulator, MBNL1, which is sequestered by expanded CUG and CCUG in DM, binds the BIN1 pre-mRNA and regulates directly its alternative splicing. Finally, reproducing BIN1 splicing alteration in mice is sufficient to reproduce the DM features of T-tubule alterations and muscle weakness. We propose that alteration of BIN1 alternative splicing regulation leads to muscle weakness, a predominant pathological feature of DM.
Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy.
Specimen part
View SamplesTREM-2 has been described to be a phagocytic receptor. We assessed the influence of TREM-2 on gene expression in alveolar macrophages (AM)
The triggering receptor expressed on myeloid cells 2 inhibits complement component 1q effector mechanisms and exerts detrimental effects during pneumococcal pneumonia.
Specimen part
View SamplesThe exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs at a late stage of the splicing reaction and remains associated following export to the cytoplasm. This complex is involved in several cellular post-transcriptional processes including mRNA localization, translation and degradation. The EJC plays an additional role in the splicing of a subset of genes in Drosophila and in human cells but the underlying mechanism remains to be elucidated. Here, we have found a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding follicular cells. This function is mediated specifically through the control of the splicing of the piwi transcript. In absence of RnpS1 one of the piwi intron is retained. This intron contains a weak 5’ splice site as well as degenerate transposon fragments, reminiscent of heterochromatic introns. In addition, we identified a small A/T rich region, which alters its polypyrimidine tract (PPT) and confers the RnpS1’s dependency. Finally, we showed that the removal of this intron by RnpS1 requires the initial splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of challenging introns following its initial deposition to adjacent exon junctions. Overall design: In total there are 4 different conditions. Comparisons were made between piwi mutant vs control piwi and rnps1 KD vs controls RnpS1
The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript.
Specimen part, Subject
View SamplesTo gain insights into the molecular pathogenesis of DCM caused by LMNA mutation, a doxycycline-inducible (Dox-Off) gene expression system was used to express either a wild type (WT) or a mutant LMNA containing the pathogenic variant p.Asp300Asn (LMNAD300N) in cardiac myocytes. The LMNAD300N is associated with DCM in patients with atypical progeroid/Werner syndrome and non-syndromic cardiac progeria. Expression of the mutant LMNAD300N protein in cardiac myocytes led to severe fibrosis, apoptosis, cardiac dysfunction, and premature death. RNA-seq was performed (prior to onset of cardiac dysfunction) to identify gene signatures and transcriptional regulators responsible for this phenotype. Mechanistic studies identified activation of E2F/TP53/DDR, as a major mechanism responsible for the pathogenesis of DCM caused by the LMNAD300N mutation. Overall design: RNA-seq analysis from 2-week old WT and mice expressing mutant LMNA (LMNAD300N)
DNA Damage Response/TP53 Pathway Is Activated and Contributes to the Pathogenesis of Dilated Cardiomyopathy Associated With LMNA (Lamin A/C) Mutations.
Cell line, Subject
View SamplesMutations in the LMNA gene causes set of disorders collectively referred to as laminopathies that include dilated cardiomyopathy. Lamin A/C proteins a components of nuclear lamina forms distinct nuclear domains called lamina associated domains (LADs). The roles of LADs in DCM is not known. To identify LADs and characterize their associations with CpG methylation and gene expression in human cardiac myocytes isolated from patients with DCM and controls we performed Chromatin immunoprecipitation-sequencing (ChIP-Seq), reduced representative bisulfite sequencing (RRBS), and RNA-sequencing (RNA-Seq) in 5 control and 5 DCM hearts with defined pathogenic variants in the LMNA gene. LADs are redistributed in DCM, are associated with CpG methylation and suppressed transcription, contributing to the pathogenesis of DCM in laminopathies. Overall design: integrated analysis of ChIP-seq for LMNA from Cardiac myocytes and RNA-seq and RRBS from 5 control and 5 DCM human heart sample
Genomic Reorganization of Lamin-Associated Domains in Cardiac Myocytes Is Associated With Differential Gene Expression and DNA Methylation in Human Dilated Cardiomyopathy.
Sex, Age, Subject
View SamplesSnail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here that Snail1 plays also a key role in tumor associated fibroblasts since is necessary for enhancement by these cells on epithelial cells tumor invasion. Snail1 expression in fibroblast requires signals derived from tumor cells such as TGF-b; reciprocally, in fibroblasts Snail1 organizes a complex program that favors collective invasion of epithelial cells at least in part by the secretion of diffusible signaling molecules, such as prostaglandin E2. The capability of human or murine tumor-derived cancer associated fibroblasts to promote tumor invasion is associated to Snail1 expression and obliterated by Snail1 depletion. In vivo experiments show that tumor cells co-transplanted with Snail1 depleted fibroblasts show lower invasion than those xenografted with control fibroblasts. Finally Snail1 depletion in mice prevents the formation of breast tumors and decreased their invasion. Therefore, these results demonstrate that the role of Snail1 in tumor invasion is not limited to its effect in EMT but dependent on its expression in stromal fibroblasts where it orchestrates its activation and the crosstalk with epithelial cells.
Snail1-Dependent Activation of Cancer-Associated Fibroblast Controls Epithelial Tumor Cell Invasion and Metastasis.
Specimen part
View SamplesGlucocorticoids (GCs) are commonly used to treat patients suffering from lymphoid malignancies i.e. leukemia and multiple myeloma. Although GCs are known to be strong inducers of apoptosis in lymphoid cells, the molecular determinants of GC therapy resistance are poorly understood. Although GC treatment triggers important changes in gene expression, few studies have addressed the regulatory role of small regulatory microRNAs (miRNAs) in GC therapy response. Only recently, aberrant microRNA expression has been linked to the development of haematological malignancies and microRNAs have become master regulators of drug resistance. We identified GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate multiple genes involved in cell cycle control, cell organization and cell death in MM1S, which remain unaffected in MM1R cells. Correspondingly, GCs selectively trigger cell death in MM1S but not in MM1R. Out of 32 microRNAs responsive to GC in MM1S cells but not in MM1R cells, mir-150 was identified as the most persistent GC responsive microRNA. Furthermore, Ingenuity Pathways Analysis (IPA) revealed that ectopic transfection of a synthetic mir-150 mimics GC therapy response in MM1S cells, associated with selective changes in mRNA levels of typical GR transactivated and transrepressed target genes. Although mir-150 largely mirrors GC responsive changes in gene expression of the transcription factor Myb, GR chaperone FKBP5, cell cycle modulator proteins (IL23A, SKP2, CDKN1A), chemokine signaling proteins (CXCR4, CX3CR1, CCL3) and mTOR/UPR stress related proteins (DDIT4, TXNIP), we also observed mir-150 selective effects on transcription factors (NR3C2 (MR), Myb, Fos, Jun, C/EBP-beta, IRF4, NFE2L1, ATF3, ATF4,), chaperone molecules HSPA8, HSP90AB1), the sodium channel SCNN1G and UPR stress proteins (TRIB3, DDIT3). Remarkably, mir-150 overexpression was not able to overcome GC therapy resistance, since we could not detect GC like effects of mir-150 in GR (NR3C1) deficient MM1R cells. Altogether GC-inducible mir-150 adds a novel complex layer of regulation for fine tuning GC specific therapeutic responses in multiple myeloma.
Ectopic microRNA-150-5p transcription sensitizes glucocorticoid therapy response in MM1S multiple myeloma cells but fails to overcome hormone therapy resistance in MM1R cells.
Cell line, Treatment
View Samples